Through-bond effects in the ternary complexes of thrombin sandwiched by two DNA aptamers
Nucleic Acids Research
Through-bond effects in the ternary complexes of thrombin sandwiched by two DNA aptamers
Andrea Pica 1 2
Irene Russo Krauss 1 2
Valeria Parente 2
Hisae Tateishi-Karimata 0
Satoru Nagatoishi 0 4
Kouhei Tsumoto 4
Naoki Sugimoto 0 3
Filomena Sica 1 2
0 Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University , 7-1-20 Minatojima-minamimachi, Kobe 650-0047 , Japan
1 Institute of Biostructures and Bioimaging, CNR , Via Mezzocannone, 16, I-80134 Naples , Italy
2 Department of Chemical Sciences, University of Naples Federico II , Via Cintia, I-80126 Naples , Italy
3 Graduate School of Frontiers of Innovative Research in Science and Technology (FIRST), Konan University , 7-1-20 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047 , Japan
4 Department of Bioengineering, School of Engineering, The University of Tokyo , 7-3-1 Hongo, Bunkyo-Ku, Tokyo 113- 8656 , Japan
Aptamers directed against human thrombin can selectively bind to two different exosites on the protein surface. The simultaneous use of two DNA aptamers, HD1 and HD22, directed to exosite I and exosite II respectively, is a very powerful approach to exploit their combined affinity. Indeed, strategies to link HD1 and HD22 together have been proposed in order to create a single bivalent molecule with an enhanced ability to control thrombin activity. In this work, the crystal structures of two ternary complexes, in which thrombin is sandwiched between two DNA aptamers, are presented and discussed. The structures shed light on the cross talk between the two exosites. The through-bond effects are particularly evident at exosite II, with net consequences on the HD22 structure. Moreover, thermodynamic data on the binding of the two aptamers are also reported and analyzed.
Simultaneous interaction of multiple ligands with
biomolecules often finely modulates biological
processes. New strategies for the design of pharmaceutical
agents could possibly take into account the synergistic
effect of polyvalent binding (1). Among others, the case
of human -thrombin (thrombin) is particularly
interesting. Thrombin plays a pivotal role in the coagulation
cascade maintaining blood hemostasis by balancing
proand anti-coagulant actions (2–4). Its activity is regulated
and controlled by the binding of several cofactors and
substrates on two electropositive surfaces, called exosite I
and II (Figure 1), which, together with the catalytic site,
make this protein unique in the pancreatic trypsin family
of serine proteases.
In particular, exosite I is involved in binding to
fibrinogen, platelet receptor PAR-1, thrombomodulin and to
endogenous and exogenous inhibitors, while exosite II
interacts with heparin, F2 prothrombin fragment and
physiological inhibitors such as antithrombin III and nexin-I (5).
Ligand binding to either exosite I or exosite II may
influence the organization of the active site and/or the reactivity
of thrombin. For these reasons, considerable efforts are
currently being made to identify effectors of the enzyme that
are able to regulate the onset and progression of
cardiovascular diseases (6).
A special class of thrombin synthetic ligands is
represented by DNA aptamers, which are DNA oligonucleotides
that bind specific target molecules (7,8). They are identified
by in vitro selection from large random sequence libraries,
through a process also known as SELEX. Several
properties of aptamers make them very attractive as therapeutic
compounds. They have little immunogenicity and a
wellestablished synthesis protocol and chemical modification
technology, which allow a fine-tuning of their bioavailability
and pharmacokinetics. They usually bind their target with
dissociation constants in the low-nanomolar range.
Moreover, complementary oligonucleotide antidotes can reverse
aptamer actions facilitating the control over their activity in
The ability of oligonucleotides to adopt different
threedimensional structures allows them to form complementary
shapes that very well fit or embrace the recognition site of
their target. However, only a few structures are available
to show how aptamers can assume complex conformations
that enable specific binding to proteins that do not normally
interact with nucleic acids (10–19).
Two DNA aptamers, HD1 (7) and HD22 (20), directed
to exosite I and exosite II, respectively, are by far the
most studied thrombin binding aptamers both for
therapeutic and for biosensing purposes (21,22). We have
recently unraveled the structures of thrombin in complex with
these aptamers showing that HD1 adopts an antiparallel
Gquadruplex architecture (23–26), whereas HD22 presents a
mixed duplex–quadruplex folding (27). Since the
interaction of each aptamer is mediated by different protein
subdomains, it is possible to enhance their activity just by
linking them together thus generating a bivalent aptamer with
improved affinity and specificity (28–38). In particular, a
relevant enhancement of functional affinity has been
obtained by using linkers based on PEG-chains (32),
randomized DNA sequences (36) or DNA weave tiles (33,37,38).
The strategy adopted to conjugate the two aptamers does
not require a detailed knowledge of the protein–aptamer
interaction at the two exosites. However, the elucidation at
the atomic level of the way in which both aptamers bind
to the protein in a ternary complex may suggest new
approaches for the design of thrombin inhibitors with
enhanced specificity. It should be noted that while the
positioning of HD1-like aptamers at exosite I is well established
(24–26,39), HD22 was found to adhere to exosite II in an
unexpected bent conformation (27), whose details could be
finely regulated by the presence of the aptamer bound at
Here we report the crystal structure of two ternary
complexes, which differ for the HD1-like aptamer bound at
exosite I. The resulting structures, which are embedded in a
different packing organization, fully confirm the
conformation of HD22 already found in the binary complex (27),
but also display small differences that can be interpreted in
term of a long-range communication between the two
exosites. Thus, the present data can represent a good starting
model for a computational analysis aimed at a better
understanding of the cooperative mechanisms generated by the
simultaneous binding of ligands at the two exosites (40–43).
Isothermal titration calorimetry (ITC) data on the
formation of the binary and ternary complexes are also reported
MATERIALS AND METHODS
Thrombin (Haemtech Technologies, Essex Junction,
VT, USA) inhibited by PPACK
(D-Phe-Pro-Argchloromethylketone) was equilibrated against a 0.75
M KCl solution using a Centricon mini-concentrator
(Vivaspin 500, Sartorius, Goettingen, Germany) and a
refrigerated centrifuge (Z216MK, Hermle Labortechnik,
Wehingen, Germany). The two HD1-like aptamers, lacking
the nucleobase of T3 ( T3) and T12 ( T12), were
synthesized as previously described (44). More information
about the sequence of both aptamers is reported in
Supplementary Data. Each aptamer was dissolved in 10 mM
sodium phosphate buffer pH 7.1 up to a concentration of
2 mM. In order to induce the correct fold of the aptamers,
a process of annealing is mandatory. Thus, both solutions
were heated for 10 min at 85◦C, slowly cooled down and
stored at 4◦C overnight.
HD22 was purchased from Sigma-Genosys and handled
in the same way. Its sequence is reported in Supplementary
Data. A solution of the aptamer at a concentration of 4
mM was prepared using 10 mM sodium phosphate pH 7.1
as buffer. An annealing process was necessary to induce its
The ternary complex was then prepared depositing a
twofold molar excess of HD22 onto a frozen sample of
thrombin. The system was left for about 3 h at 4◦C. Then
the solution was frozen again and a 2-fold molar excess of
the T3 (or T12) solution was deposited onto it. The
system was let equilibrating at 4◦C overnight. The final
solution was then diluted and extensively washed to remove the
excess of the aptamers and was finally concentrated to about
0.2 mM. The final buffer was changed to 25 mM sodium
phosphate buffer pH 7.1, 0.1 M NaCl.
Crystallization and data collection
Crystallization conditions for the two complexes were
identified after extensive screening of sitting-drop
crystallization experiments at 20◦C in 96-well plates (Greiner Bio-One,
Monroe, NC, USA) using an Automated Protein
Crystallization Workstation (Hamilton Robotics) and precipitant
solutions of commercially available crystallization screens
(Hampton Research Crystal Screen 1, Crystal Screen 2 and
Crystals suitable for X-ray diffraction data collection
were obtained after very fine optimization of the
crystallization conditions in the hanging drop vapor diffusion method
mixing 0.5 l complex solution with 0.5 l reservoir
solution at 20◦C. Crystals of the ternary complex of
thrombin with HD22 and T3 grew in 28% MPEG 2000, 0.1 M
Bis/Tris pH 6.5 and belong to space group I23. Crystals
of the ternary complex of thrombin with HD22 and T12
grew in 18% PEG 3350, 0.2 M sodium formate, acetonitrile
2% v/v and belong to space group C2.
Crystals were then cryoprotected with 20% (v/v)
glycerol and flash-cooled in liquid nitrogen. Data were
collected at the CNR Institute of Biostructures and Bioimages,
Naples, Italy, using a Saturn944 CCD detector equipped
with CuK X-ray radiation from a Rigaku Micromax 007
HF generator. Datasets were processed using HKL2000
software (45). Matthews coefficient calculations suggested
in both cases the presence of a 1:1:1 ternary complex in the
asymmetric unit. Detailed statistics of data collection are
reported in Supplementary Table S1.
Structural solution and refinement
The phase problem was solved by molecular replacement
using PHASER (46). Thrombin heavy and light chains
and HD22 from PDB structure 4I7Y, and HD1 variants
from 4LZ1 and 4LZ4 were used as independent search
models. Crystallographic refinement was performed using
PHENIX (47) and REFMAC5 (48), while manual model
building was handled with COOT (49). After the inclusion
of low-resolution data and bulk solvent correction, the final
R/Rfree values were 15.5/20.8 for Ter T3 and 20.8/26.8
for Ter T12. The structures were analyzed using
MolProbity (50) and NucPlot (51). A summary of refinement
statistics is show in Supplementary Table S1. Molecular
graphics figures were prepared with PyMOL (DeLano Scientific,
Palo Alto, CA, USA).
ITC measurements and binding assays
Binding energies for the interactions between aptamers and
thrombin were determined in a buffer of 25 mM sodium
phosphate (pH 7.1), 100 mM NaCl using ITC on a
MicroCal iTC200 (GE Healthcare, Tokyo, Japan). Degassed
thrombin solutions (4-5 M) were titrated with each
aptamer solution (40–50 M). Control experiments were
carried out to calculate the heat of dilution for aptamers. The
thermogram for the interaction was determined by
subtracting the heats of sample experiments from those of the
control experiment. The thermograms were fitted to
obtain the KA value analyzed with Origin 7 software
(MicroCal Inc., Northampton, MA, USA) for models that assume
a single set of identical binding sites. Thermodynamic
parameters governing protein–aptamer interactions were
estimated by the following standard relationships [Equations
(1) and (2)]:
G = − RT ln (KA)
H − T
where G is the binding free energy change, H is the
binding enthalpy change, S is the binding entropy change, R is
the gas constant, and T is the temperature in Kelvin.
Figure 2. Surface representation of Ter T3 and Ter T12. The main
difference is the orientation of the HD1 variant (in green) at exosite I. It can
easily be seen that the TGT loop of HD1 points down in Ter T3 and up
in Ter T12.
careful set-up of experimental conditions is always crucial
(52) but sometimes it is just not enough. In particular, our
attempts to crystallize a ternary complex between
thrombin, HD1 and HD22 have not been successful despite the
hundreds of crystallization trials. On one side, the
flexibility of the nucleic acid chains and, on the other, the two
modes of binding of HD1 (26) make the solution a
heterogeneous mixture of species, thus hampering the
crystallization process. In order to overcome this problem,
crystallization trials were performed using either one of two HD1
variants, named T3 and T12, which lack the T3 or T12
nucleobase, respectively (see Supplementary Data and Figure
S1). Indeed, in a previous paper (26) we have shown that the
removal of one base from a TT loop also removes the
degeneracy of the two HD1 binding modes. Indeed the use of the
HD1 variants was successful; the two ternary complexes,
hereafter referred to as Ter T3 and Ter T12, formed
ordered and well-diffracting crystals suitable for X-ray
diffraction data collection (Supplementary Figure S2).
Crystallization of the two ternary complexes
To our knowledge, the crystallization of a protein in
complex with two different DNA aptamers has never been
accomplished. In order to succeed in this task, an extremely
Overall crystal structure of the ternary complexes
The structure of the two complexes was solved at 2.95 and
3.58 A˚ resolution with Rfactor/Rfree values of 0.155/0.208
and 0.208/0.268 for Ter T3 and Ter T12, respectively.
Figure 3. (A) Schematic representation of cyclic H-bond pattern between
residues Arg75, T4, Arg77A and T13. The arrangement stack onto a
Gtetrad and is characterized by two cation- /H-bond stair motifs (53). (B)
View of the hybrid quadruplex with the protein–DNA tetrad highlighted
Statistics of data collection and refinement for both
complexes are reported in Supplementary Table S1
In both cases, HD22 binds thrombin exosite II, while the
HD1 variant binds exosite I on the opposite side of
thrombin molecule. A surface representation of the two complexes
is shown in Figure 2. The two ternary complexes present
a similar architecture, with the main structural differences
arising from the orientation of the HD1 variant: with
respect to the protein T3 and T12 are related by a 180◦
rotation around the quadruplex axis of the aptamer. Due
to the higher resolution of crystallographic data of Ter T3
than Ter T12, hereafter we will mainly refer to Ter T3.
Protein heavy chain (residues 16–246) and light chain
(residues 1B–14J) are well defined in the electron-density
maps as well as PPACK into the active site and the sugar
at the glycosylation site, with the only exception of the
disordered autolysis loop (residues 146–150). In comparison
to the aptamer-free protein (PDB code: 1PPB) the rmsd,
after the superposition of all the C protein atoms, is 1.0
A˚, very close to the values found for thrombin/aptamer
binary complexes (26,27). The two aptamers bury a very large
protein surface area (Table 1).
HD1 variants and exosite I
The HD1 variants have the canonical antiparallel
Gquadruplex architecture that coordinates a sodium ion at
the center of the G-core. It presents only minor differences
with that found for the binary complexes, in which the
quadruplex motif coordinates a potassium ion. The highly
flexible TGT loop, which is placed far away from the protein
interface, lacks tight packing interactions and is partially
In both complexes the TT and TN loops (N stands for the
DNA spacer, replacing T3 and T12 in T3 and T12
variants, respectively) of the HD1 variants grab a protruding
region of exosite I: the TT loop interacts with the A-region of
exosite I (Arg75, Glu77, Arg77A, Asn78 and Ile79) whereas
the defective TN loop interacts with the B-region (Arg75
and Tyr76) (Supplementary Figure S3).
Thus in the two variants the aptamer is rotated by 180◦
around the pseudo molecular twofold axis normal to the
quadruplex structure: this operation interchange the TT
loop with the TN loop and allows the TT loop to interact in
both cases with the protein A-region; the TGT loop also
acquires a different orientation. These findings strengthen our
previous suggestion that the interaction of the TT loop with
the A-region of the protein is the most effective for the
stabilization of the complex (26). The interface of thrombin with
HD1 variants involves four bases, for a total of about nine
H-bonds. One important interaction for the recognition
process (26) involves residue Glu77 and the first thymine of
the TT loop (T3 in Ter T12, T12 in Ter T3).
The guanidinium groups of Arg75 and Arg77A and the
nucleobases of the loop residues T4 and T13 are linked
together by a cyclic network of hydrogen bonds in a roughly
planar arrangement (Figure 3A). This hybrid tetrad formed
by both protein and oligonucleotide residues stacks onto
the guanines of the first tetrad (G2-G5-G11-G14) at an
average distance of about 3.4 A˚ . The interactions of the
two Arg residues are typical cation- /H-bond stair motifs
(53); all together, these interactions build up a peculiar
Gquadruplex (Figure 3B).
HD22 and exosite II
All residues of HD22 could be fitted in the electron
density maps except for the 5 terminal guanine residue of
Ter T3 and for both terminal residues of Ter T12. The
overall structure of HD22 (Figure 4A) is very similar for
the two complexes (the rmsd is 0.98 A˚ ), and presents
interesting new features with respect to the
characteristic mixed duplex/quadruplex architecture highlighted by
Russo Krauss et al. for HD22 (27) in the binary complex
(the rmsd is 1.24 A˚ for Ter T3 and 1.39 A˚ for Ter T12).
Similarly to the binary complex the two tetrads
G8-G11G17-G20 (tetrad I) and G5-G7-G12-G16 (tetrad II) of the
quadruplex domain stack on top of each other to form
the eight-guanine core. The two tetrads are spaced by four
loops, which contribute to the stability of this local
conformation by forming two inter-loop Watson–Crick (WC) base
pairs that stack on the guanine residues of the G-tetrads.
The duplex domain presents the same three canonical
WC base pairs (T2-A26, C3-G25 and C4-G23) found in the
binary complex. Moreover, the transition from the duplex
to the quadruplex region occurs in one strand without
intermediate residues, whereas in the second strand it is
mediated by two residues, G21 and G22. The former (G21) forms
two strong hydrogen bonds with the phosphate group of G5
(G-fork). The bulging out of the two residues forces the
duplex axis to adopt an almost orthogonal orientation with
the quadruplex axis (Figure 4A), thus creating an
interaction surface contributed by the quadruplex and duplex
motifs that extensively covers exosite II of the protein (Figure
The protein–aptamer interactions provide a structural
basis for the interpretation of the mutagenesis experiments
performed by Mayer and collaborators (54), which could
not be explained on the basis of the previous model (20).
Indeed, the complete loss of binding activity resulting from
the mutations T6C, G8A, G23A can be explained by the
fact that T6, G8, G23 are all involved in key intramolecular
interactions that would be disrupted upon the substitution.
On the other hand, the lack of any effect on the binding for
the mutation G22A is expected. Although G22 is involved
in stacking interactions with the adjacent residues G21 and
G23 and is hydrogen bonded to the nearby Arg233, both
these interactions would remain basically unchanged upon
Abbreviations: N, binding stoichiometry of oligonucleotides to thrombin; KA (107 M−1), binding constant; H, −T S and G (kcal mol−1) changes in binding enthalpy,
binding entropy and binding free energy at 25◦C, respectively.
mutation with an adenine (see also Supplementary Data for
Despite the close similarity in the overall organization of
HD22 with respect to the binary complex, the present data
show new interesting details that throw further light on the
flexibility and adaptability of the aptamer structure. Indeed,
in the ternary complexes a sodium ion is present between
the two tetrads (Figure 4C). The ion is 2.6 A˚ from the least
square plane through the bases of tetrad II and only 0.9 A˚
from that of tetrad I. The bases G8-G11-G17-G20 present
the classical anti-syn-anti-syn conformation sequence, and
a regular arrangement of Hoogsteen-like H-bonds
(Supplementary Figure S4). The four O6 atoms point toward the
quadruplex axis and the cation coordinates the O6 atoms at
a distance of 2.1 A˚ in a square pyramidal geometry. Tetrad
I and tetrad II are more planar with respect to the binary
complex, where their puckering decreases the space
available for the coordination of the cation. Moreover, the four
bases of tetrad II, which in the binary complex show the
non-canonical anti-syn-anti-anti conformational sequence
and a unique H-bond pattern, also have a non-canonical
conformation with all residues in the anti conformation.
In this novel organization the four nucleobases direct
alternatively N2 and O6 toward the quadruplex axis forming
a cyclic pattern of hydrogen bonds (Supplementary Figure
Figure 6. Scheme of all reactions studied by ITC. For each reaction,
thermodynamic parameters ( H, −T S and G) have been calculated and
are reported in Table 2.
The tertiary structure of the molecule opens a
throughspace communication pathway between the duplex and
quadruplex regions via the G-fork. This pathway is clearly
elucidated by the present structures. Indeed, the modified
base orientations and hydrogen bond scheme of tetrad II
causes through the G-fork a small reorganization at the
C3G25, and C4-G23 region of the duplex (Figure 5). This
results in a slight contraction of the quadruplex–duplex
mouth, which allows a better bite of the aptamer on exosite
In the correspondence to the shift of the duplex moiety,
correlated movements are also observed on the protein side.
In particular, a small rotation of the external helical region
125–129 and a shift of the loop 162–182, which form a cavity
on the protein surface, allows a better fit of the T24
backbone. This residue, which bulges out from the duplex strand
and is an anchor point of HD22 on thrombin surface, moves
about 1.5 A˚ more deeply buried inside the cavity (Figure
5). With respect to the binary complex additional contacts
between the double helix of HD22 and the thrombin
surface are also observed (Supplementary Table S2). At the
interface between the quadruplex region and the protein,
the modifications are less evident and most of the
interactions present in the binary complex are generally well
preserved. Despite their completely different packing
organization, the above differences are common to the ternary
complexes Ter T3 and Ter T12, suggesting that they are not
merely an artifact of the crystal packing. Interestingly, they
are observed in the presence of PPACK bound at the active
site that is known to reduce the protein flexibility (55). It
should be noted, however, that the protein maintains some
degree of flexibility even in the active-site liganded form
ITC data on DNA aptamer binding
In addition to the structural data, thermodynamic
parameters for the binding of the aptamers to human thrombin
have been obtained by ITC analysis (Supplementary
Figure S4), according to the scheme reported in Figure 6.
Experiments have been performed at 25◦C using high purity
thrombin samples with the active site covalently inhibited
by PPACK, in order to minimize possible artifacts caused
by the autocatalytic degradation of the enzyme. In
agreement with previous suggestions (57,58), the results strongly
indicate that all three aptamers (HD1, T3 and HD22)
form with thrombin a complex with a 1:1 stoichiometry
(Table 2 and Figure 6A–C). The H and −T S values for
thrombin and T3 were similar with those for thrombin
and HD1 indicating that the loss of the - stacking
interactions between Tyr76 and the missing nucleobase did not
significantly affect the change of H and −T S. As far as
HD22 is concerned, the finding that the enthalpy
contribution to the binding is smaller than that observed in the case
of the interaction of HD1 is unexpected. Indeed, the value
of the aptamer/thrombin contact area is doubled in the case
of HD22 with respect to that found for HD1 (Table 1) (27).
The smaller value of H, however, could be the result of
the conformational flexibility of HD22 in solution, which
should be much higher than that of HD1. Some
conformers have to change structure to be able to bind exosite II
with probably an enthalpy penalty. On the other hand, the
low value of −T S is likely associated to a more substantial
dehydration occurring with the binding of HD22 compared
to the binding of HD1. Indeed, the binding site at exosite
II of HD22 is twice as large as the binding site at exosite
I of HD1, it is richer in polar charged residues and more
densely hydrated. It is worth noting that G is practically
unchanged with respect to HD1 or T3 complexes.
Finally, it has to be underlined that the present study
represents the first complete comparative thermodynamic
analysis of the binding of each aptamer alone as well as of
the binding of each aptamer when the other exosite is
occupied (Table 2 and Figure 6D-G). It should be noted that
these thermodynamic parameters do not manifest the
presence of exosite-exosite interactions, which have been clearly
evidenced in literature by other methods (40–43).
The existence of two binding sites on the surface of
thrombin allows protein activity to be finely regulated; indeed,
recent data on the allosteric interplay among thrombin
functional sites (the two exosites and the catalytic site) have
shown that the simultaneous presence of HD1 and HD22
significantly enhances thrombin-catalyzed hydrolysis of a
peptide substrate (59). However in the ternary complexes,
whose structures we report here, the HD1-like aptamer
located at exosite I and HD22, the mixed duplex/quadruplex
aptamer that binds at exosite II, are more than 30 A˚ apart,
too far away for a through-space interaction between them
to occur. Moreover, the thermodynamic data do not
indicate the presence of significant cooperative effects in the
binding of the two aptamers. Nonetheless, the structural
details of the ternary complexes in comparison to the
corresponding binary ones (26,27) indicate that, at least for
HD22, small but intriguing differences are observed. In
particular, the reorganization of tetrad II with a different
orientation of G12 and the more massive presence of the sodium
ion, which is present only at a very low level, if any, in the
binary complex, slightly displaces the duplex region and
causes the whole aptamer to improve the adhesion to exosite
II. Alternatively, it can be said that small modifications at
the protein/HD22 interaction surface may ultimately
influence the reorganization of the quadruplex region. We
surmise that this may be due to the binding of the ligand at
exosite I that is likely to modify the dynamics of the whole
Incidentally, also in the thrombin-HD22 binary complex
crystallized in the presence of K+, the ion is not present at
an appreciable level in the quadruplex moiety (data not
A through-bond propagation effect has already been
suggested on the basis of various studies (60–62). The results
indicate that the binding of a ligand at one site reduces the
dynamics of the whole protein so that a smaller number of
conformations are accessible for the binding of a second
ligand (62–64). The crystallographic data reveal that the
binding of HD22 at exosite II does not produce visible effects
on the binding of the HD1-like aptamer at exosite I. Vice
versa, the presence of a ligand at exosite I causes small but
significant effects for the binding of HD22. With respect to
the former, this aptamer is endowed with a much higher
conformational flexibility due to its composite nature. In
particular, the bent active conformation of HD22 displays
intriguing intramolecular duplex–quadruplex interactions.
The protein surface covered by HD22 is much broader than
that covered by HD1 and includes the C-terminus of the
heavy chain and residues 96–97A that were found to be very
dynamic by nuclear magnetic resonance and molecular
dynamics methods (56). This peculiarity, in addition to the
bimodular nature of HD22 and to the role played by the
sodium cation may render this aptamer more prone to
reveal the subtle effects of the protein dynamics and the
perturbation exerted by the presence of a ligand at exosite I.
The two structures reported in this paper represent the first
example of a protein simultaneously bound to two aptamers
in a ternary complex. In particular, the case of thrombin
represents an intriguing example of how ligand binding to
more than one exosite can control enzymatic activity.
In the structures of the two ternary complexes, the
presence in the protein active site of PPACK, which was used
to avoid the heterogeneity of protein solution induced by
autoproteolysis, stabilizes the functional core of the protein
and reduces the backbone dynamics (55). This prevents a
marked structural identification of an interplay between the
active site and the two exosites. However, few clues
regarding site–site interaction clearly emerge from our data and
can be a useful starting point for the computational analysis
of dynamics and long-range effects connected to the
simultaneous binding at the two exosites, a question that remains
still open and controversial. Furthermore, although various
bivalent aptamers have been already produced, we believe
that the present structures may have direct implications on
novel strategies to design direct thrombin inhibitors with
Coordinates and structure factors for Ter T3 and Ter T12
have been deposited in the Protein Data Bank under ID
codes 5EW1 and 5EW2, respectively.
Supplementary Data are available at NAR Online.
Giosue` Sorrentino and Maurizio Amendola (Institute of
Biostructures and Bioimaging, CNR, Naples, Italy) are
gratefully acknowledged for technical assistance during
Grants-in-Aid for Scientific Research from a Ministry
of Education, Culture, Sports, Science and Technology
(MEXT) (in part); MEXT-Supported Program for the
Strategic Research Foundation at Private Universities
(2014-2019), Japan; Hirao Taro Foundation of KONAN
GAKUEN for Academic Research; Okazaki Kazuo
Foundation of KONAN GAKUEN for Advanced Scientific
Research; Chubei Itoh Foundation. Funding for open access
charge: Grants-in-Aid for Scientific Research from a
Ministry of Education,Culture, Sports, Science and Technology
Conflict of interest statement. None declared.
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